D-2-deoxyribose-5-phosphate aldolase (EC4.1.2.4) (hereinafter, referred to as “DERA” for short) is a generic name for an enzyme which catalyzes aldol condensation which synthesizes D-2-deoxyribose-5-phosphate from glyceraldehyde-3-phosphate and acetaldehyde as substrates, and a reverse reaction thereof (retro-aldol reaction). Reactivity of Escherichia coli (E. coli)-derived DERA was analyzed in detail, and reported to have a relatively wide spectrum of substrate-specificity, thus catalyze aldol condensation of various aldehydes to produce chiral hydroxyaldehydes (See Non-Patent Document 1).
As shown in the reaction scheme of FIG. 1, it is reported that in the aldol condensation of various aldehydes with acetaldehyde, a compound having the number of carbon atoms increased by four compared to that of the starting aldehyde is obtained by subjecting a hydroxyaldehyde, which is resulted from aldol condensation of a single molecule of aldehyde and a single molecule of acetaldehyde, to further aldol condensation with another single molecule of acetaldehyde. It has been reported that this compound resulting from aldol condensation with two molecules of acetaldehyde is produced as the main reaction product because the compound takes a stable lactol structure (See Non-Patent Document 2).
However, the conventionally known activity of DERA to various aldehydes as described above is reported to be extremely low, and to be less than 1/100 of the activity with respect to glyceraldehyde-3-phosphate and acetaldehyde, which are the original substrates (See Non-Patent Document 3). It is also reported that aldehydes are strong inhibitors to enzymes, and DERA is also inhibited by aldehydes (See Non-Patent Document 4). Accordingly, aldol condensation of these aldehydes requires a large amount of the enzyme. For example, Patent Document 1 described above discloses the synthesis of a 2,4,6-trideoxyhexose derivative via a 4-substituted-3-hydroxybutylaldehyde intermediate, wherein an increased amount of D-2-deoxyribose-5-phosphate aldolase to the degree of 125 U/mmol to 150 U/mmol was added, relative to the total number of moles of the substrates, namely, acetaldehyde and substituted acetaldehyde. Similarly, it is believed that even the reaction of condensation one acetaldehyde molecule requires the addition of an increased amount of the enzyme to the degree of 80 U/mmol to 100 U/mmol. Furthermore, for the purpose of alleviating inhibition, multiple addition of aldehydes and DERA is also being investigated (See Patent Document 2).
The aldol condensation reaction by DERA has another problem in controlling the number of acetaldehyde molecules to be condensed. Although the attempts of changing the concentration ratio of two aldehydes subjected to the aldol condensation or changing the amount of enzyme have been made, it has been difficult to control the number of acetaldehyde molecule, only except for the case of aldol condensation of several aldehydes having hydroxyl groups on the α-position or β-position, with acetaldehyde (See Non-Patent Document 5, Non-Patent Document 6 and Patent Document 3). Due to these problems, it has been difficult to practicalize the aldol condensation by DERA.
From the viewpoint of highly stable DERA, a thermally stable DERA which is derived from a hyperthermophilic bacterium, Aeropyrum pernix, has been reported (See Patent Document 4). This enzyme is reported to have high thermal stability and high stability against polar organic solvents such as methanol, ethanol and the like (See Non-Patent Document 8). Additionally, DERA derived from thermophilic bacteria such as Thermotoga maritima, Thermus thermophilus and the like have been reported, but nothing is reported concerning the stability against aldehydes, activity on aldol condensation or the like of these thermophilic bacterium-derived DERA (See Non-Patent Document 9).
In general, a thermophilic bacterium-derived enzyme exhibits high activity in a high temperature region near the original growth temperature, and has significantly low activity in the normal temperature region. In the case of the aldol condensation in which a highly reactive compound such as aldehyde is used as the substrate, a reaction in a high temperature region is predicted to be accompanied by various side reactions. Thus, it is necessary to perform the aldol reaction under low temperature conditions, and therefore, it is generally inconceivable to use a thermophilic bacterium-derived DERA for the reaction. Furthermore, it was difficult to deduce that the enzyme would have high stability against aldehydes, merely from the fact that the enzyme has high thermal stability, since aldehydes have a property of being reactive to the lysine residue or the like that are present in proteins.
[Patent Document 1] U.S. Pat. No. 5,795,749
[Patent Document 2] WO 03/006656
[Patent Document 3] WO 03/077868
[Patent Document 4] JP-A No. 2003-250553
[Non-Patent Document 1] J. Am. Chem. Soc., Vol. 112, pp. 2013-2014 (1990)
[Non-Patent Document 2] J. Am. Chem. Soc., Vol. 116, pp. 8422-8423 (1994)
[Non-Patent Document 3] J. Am. Chem. Soc., Vol. 114, pp. 741-748 (1992)
[Non-Patent Document 4] PNAS, Vol. 101, pp. 5788-5793 (2004)
[Non-Patent Document 5] J. Am. Chem. Soc., Vol. 117, pp. 3333-3339 (1995)
[Non-Patent Document 6] Angeu. Chem. Int. Ed., Vol. 39, pp. 1352-1374 (2000)
[Non-Patent Document 7] J. Am. Chem. Soc., Vol. 114, pp. 741-748 (1992)
[Non-Patent Document 8] J. Biol. Chem., Vol. 278, pp. 10799-10806 (2003)
[Non-Patent Document 9] J. Mol. Biol., Vol. 343, pp. 1019-1034 (2004)